Figure 4.

Dual immunofluorescence for K63-ubiquitin and CDH1. K63-ubiquitin is localized as intraepithelial accumulations in the airway epithelium. The accumulations are associated with diacetyl exposure and are more frequently identified in bronchi than terminal bronchioles. A: Photomicrograph of immunofluorescence double label for K63-ubiquitin (red) and CDH1 (green) in the airway epithelium of the lung of a male Dcxr knockout mouse exposed to 200 ppm diacetyl. K63-ubiquitin forms large puncta similar to those identified by staining for total ubiquitin. B: Morphometric quantification of K63-ubiquitin positive cells in the epithelium of terminal bronchioles of wild-type (open bars) and knockout (closed bars) mice. K63 ubiquitin accumulations were only significantly increased in terminal bronchioles of wild-type mice inhaling 100 ppm. C: Morphometric quantification of K63-ubiquitin positive cells in the epithelium of bronchi. K63-ubiquitin puncta were rare except in diacetyl-exposed mice, and cells containing puncta were significantly increased relative to controls at all exposure concentrations. Knockout mice (closed bars) were significantly more susceptible than wild-type mice (open bars) at 200 ppm when the sexes were pooled for the statistical analysis. ^∗P < 0.05, ^∗∗∗P < 0.001 compared with air controls of the same genotype; ^†P < 0.05 compared with wild-type mice. Scale bar = 20 μm (A).